SUMMARY The goal of this project is to identify and characterize evolutionarily conserved host genes that are important for virus infection in organisms from humans to nematodes by exploiting a unique C. elegans virus infection system. C. elegans has played a key role in many fundamental discoveries in biology, but its use in the study of viral pathogenesis has been limited by the lack of known viruses capable of naturally infecting C. elegans. The recent discovery of Orsay Virus (ORV), the first and to date only known virus of C. elegans, provide a novel route for genetic dissection of host factors essential for virus infection. In preliminary data, a chemical mutagenesis screen in C. elegans was developed that identified two host genes essential for ORV infection. The first gene, sid-3, encodes a non-receptor tyrosine kinase that is orthologous to the human ?activated Cdc42 associated kinase? (ACK1/TNK2). The second gene, viro-2, encodes an ortholog of human Wiskott Aldrich syndrome proteins (N-WASP). ORV is a non-enveloped positive sense RNA virus; among viral families with human pathogens, ORV is most closely related to the Picorna-, Calici- and Astroviridae. Deletion of either TNK2 or N-WASP in human A549 cells led to ~1000-fold reduced titers of encephalomycarditis virus (EMCV). ~100-fold reduction in coxsackie virus B3 (CVB3) was also observed in TNK2 deleted cells. Strikingly, N-WASP is a known substrate for the kinase activity of TNK2, suggesting that these genes may comprise a key pathway utilized by viruses. In support of this possibility, a third gene in C. elegans, nck-1, which is the ortholog of the human NCK protein that binds to both TNK2 and N-WASP, was also found to be essential for ORV infection. TNK2 has not been previously implicated in virus infection and represents a novel mammalian pro-viral host factor discovered by genetic screening in C. elegans. The aims are: 1) Utilize C. elegans as a discovery engine to comprehensively identify and characterize host factors critical for ORV infection. 2) To gain mechanistic insight into TNK2 and N-WASP, the stage of EMCV and CVB3 infection impacted by these genes and their essential protein domains and substrates required for their pro-viral functions will be defined. In addition, among the C. elegans candidate genes identified in Aim 1 that have human orthologs, a subset that phenocopy sid-3 and viro-2, which are the most likely candidates to act in the same pathway, will be prioritized for similar analyses. 3) Finally, the effect of TNK2 deletion in mice on EMCV and CVB3 infection will be defined. This proposal will yield novel insights into fundamental host genes and pathways necessary for virus infection which could be novel targets for antiviral development.